Copper(2+), bis[N-[amino(imino-.kappa.N)methyl]urea-.kappa.O]-, nitrate (1:2)

Endpoint summary

Description of key information

Acute oral toxicity: 300 mg/kg < LD50 < 2000 mg/kg, OECD guideline 423, GLP compliant
Acute inhalation toxicity: data waiving (study scientifically unjustified), very low  vapour pressure (0.0000009 Pa ) and only 10 % of particles are below 13.5 µM (more than 90 % of particles are non inhalable).
Acute dermal toxicity: LD50 > 2000 mg/kg, OECD guideline 402, GLP compliant

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05/10/12 to 10/02/12

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

study performed in accordance to OECD guideline 423 and GLP except for the analysis of CuGUN. Analysis of CuGUN was performed by the chemical analysis unit of INERIS wich is not included in the GLP area of expertise of the test facility. However, this analytical phase was performed according to INERIS recorded and validated procedures, and was inspected by the Quality Assurance Personnel of the test facility for both the technical phase and the raw data generated. As a consequence, due to the approach used for control of these data, it was considered that the non-GLP analytical monitoring of the test item did not affect the study compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

yes

Remarks:

some temperature and hygrometry deviations were observed. Based on animal clinical observation, the study director concluded these deviations did not have any significant impact on the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

except analysis of the test item (see rationale for reliability)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 230g (group 300mg/kg) and 236g (group 2000mg/kg)
- Fasting period before study: approximately 5:00 pm on day before treatment to 3 hours after treatment
- Housing: in polycarbonate cages containing dust-free bedding (sawdust + chips), cages were changed at least once a week
- Diet (e.g. ad libitum): ad libitum except during fasting period, rat/mice altromin 1320, Genestil
- Water (e.g. ad libitum): ad libitum, filtered (0.2 µm) tap water
- Acclimation period: 5 to 19 depending on treatment session

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 45% to 65%
- Air changes (per hr): 15 to 20 cycles
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 2012-5-16 (first exposure) To: 2012-6-07 (last necropsy)

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

filtered tap water

Details on oral exposure:

The vehicle used in the study was selected from the results of solubility and homogeneity assays performed prior to the main test. A homogeneous suspension was obtained with filtered tap water at concentrations of 30 g/L and of 200 mg/L of CuGUN

MAXIMUM DOSE VOLUME APPLIED: 10ml/kg (max 2.4 ml /boby weight 240g)

DOSAGE PREPARATION (if unusual): CuGUN preparations, administered as a homogeneous suspension, were prepared approximately within 1h00 of dosing. CuGUN was weighted and diluted with the filtered tap water volume to obtain the desired concentration. CuGUN preparations were stirred continuously, in order to maintain a homogeneous suspension, throughout the administration period.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose:
the initial dose of 300mg/kg was chosen in accordance wit the OECD No.423 guideline
a higher dose (2000mg/kg) was subsequently selected based on the absence of mortality observed in the rats treated with the initial dose

Doses:

Animals were treated at the following doses: 297 ± 4 mg/kg and 1,996 ± 21 mg/kg

No. of animals per sex per dose:

6

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations: once on the day of arrival, once daily during the acclimation period except during the week-end, several times on the day of treatment (D0): at least once during the first 30 minutes after administration, at least 2 times during the following 4 hours, and at least once thereafter, daily from D1 to D14 (once or twice per day).
- Frequency of weighing: once on the day of arrival, once on the day of selection, once on day -1 (before fasting period), once on day 0, before treatment, once daily from D1 to D14.
- Necropsy of surviving animals performed: yes
- Other examinations performed: clinical signs, body weight, macroscopic observation of thoracic area organs (respiratory tracts (trachea, lungs), the tracheo-bronchial lymph nodes, heart, and oesophagus without opening of the tube) and abdominal organs (liver, spleen, pancreas, digestive tract (without opening of the tube, except stomach), adrenal glands, kidneys, bladder, liver and kidneys lymphatic ganglia, lumbar aortic lymphatic ganglia and female reproductive organs), histopathology of organs with macroscopic abnormalities

Statistics:

Statistical analysis was performed on body weight data (body weight and body weight change) using the GraphPad Prism® software. Assuming that body weight data are normally distributed, an ANOVA test was applied and a Bonferroni's posthoc analysis was chosen to compare daily body weight means between group 2 and group 3.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 300 - < 2 000 mg/kg bw

Mortality:

2 rats, treated with 2,000 mg/kg, were found dead at day 1 (1 rat) and at day 3 (1 rat). 2 rats, treated with 2,000 mg/kg, were euthanized because they presented signs of suffering and important weight loss approximately 20% of total weight measured before the treatment.

Clinical signs:

other: No clinical signs were observed on rats treated with the test item at 300 mg/kg, during the observation period. All rats treated with the test item at 2,000 mg/kg, presented an excessive salivation, for 10 or 30 minutes after treatment. The rat treat

Gross pathology:

Macroscopic changes considered to be related to the treatment of CuGUN were observed by external examination and in stomach, in small and large intestine, in rectum and in kidneys in rats treated with 2,000 mg/kg dose which died before the end of the study. Brown nose (2 rats), brown contour of the eyes (1 rat), soiled urogenital area (1 rat) and soiled blue green anogenital area (1 rat) were observed by external examination. Stomach presented mucosal red spots/areas (4 rats), was distended (1 rat), was filled with dark or black/blue colour content (3 rats). Small and large intestine were normal but empty of faecal matter (2 rats) and filled with black mucous (1 rat) or gray liquid content (1 rat). Some black faeces were found in rectum of one rat. Kidneys presented some dark spots (1 rat).

Other findings:

Microscopic changes considered to be related to treatment were restricted to stomach (3 rats), forestomach (1 rat), kidneys (1 rat) and thymus (1 rat) from rats treated with 2,000 mg/kg of CuGUN which died before the end of the study. It consisted in degeneration/necrosis/apoptosis in the stomach/forestomach, lymphocytolysis in the thymus and tubular degeneration/regeneration with crystal deposition in the kidneys.

Dose preparation:

Date (YYYY-mm-dd)	test item mass	total mass of the preparation	concentration	animal group
2012 -05 -16	3.00 g	100.00 g	30.0 g/L	2 (step 1)
2012 -05 -21	3.01 g	100.01 g	30.1 g/L	2 (step 2)
2012 -05 -24	20.00 g	101.13 g	200.0 g/L	3 (step 1)
2012 -05 -30	20.01 g	100.01 g	200.1 g/L	3 (step 2)

Dose administration, mortality and clinical signs: individual data

animal group	animal	Body weight at day 0	administered volume	Test item concentration	administered dose	mortality	clinical signs
2 (step 1)	161	210.7 g	2.1 ml	30.0 g/L	299.0 mg/L	scheduled death at day 14	no clinical signs observed
2 (step 1)	162	203.4 g	2.0 ml	30.0 g/L	295.0 mg/L	scheduled death at day 14	no clinical signs observed
2 (step 1)	163	213.1 g	2.1 ml	30.0 g/L	295.6 mg/L	scheduled death at day 14	no clinical signs observed
2 (step 2)	164	225.9 g	2.2 ml	30.1 g/L	292.2 mg/L	scheduled death at day 14	no clinical signs observed
2 (step 2)	165	220.5 g	2.2 ml	30.1 g/L	299.4 mg/L	scheduled death at day 14	no clinical signs observed
2 (step 2)	166	217.9 g	2.2 ml	30.1 g/L	303.0 mg/L	scheduled death at day 14	no clinical signs observed
3 (step 1)	167	221.6 g	2.2 ml	200.0 g/L	1,985.6 mg/L	found dead at day 1	excessive salivation during 10 minutes after treatment
3 (step 1)	168	210.9 g	2.1 ml	200.0 g/L	1,991.5 mg/L	scheduled death at day 14	-excessive salivation during 30 minutes after treatment-digestive disorder: soft and blue faeces
3 (step 1)	169	228.0 g	2.3 ml	200.0 g/L	2,017.6 mg/L	scheduled death at day 14	-excessive salivation during 30 minutes after treatment-digestive disorder: soft and blue faeces
3 (step 2)	170	224.2 g	2.2 ml	200.1 g/L	1,963.0 mg/L	euthanized at day 4	-excessive salivation during 30 minutes after treatment-digestive disorder: absence of faeces-suffering signs: eyelids partially closed, piloerection, round back, loss of weight
3 (step 2)	171	228.0 g	2.3 ml	200.1 g/L	2,072.1 mg/L	euthanized at day 4	-excessive salivation during 30 minutes after treatment -digestive disorder: liquid and blue faeces-suffering signs: eyelids partially closed, piloerection, round back
3 (step 2)	172	240.2 g	2.4 ml	200.1 g/L	1,998.8 mg/L	found dead at day 3	-excessive salivation during 30 minutes after treatment -digestive disorder: liquid blue faeces, soiled area: anus blue and green-suffering signs: eyelids partially closed, piloerection, round back, loss of weight

Interpretation of results:

Category 4 based on GHS criteria

Remarks:

Migrated information

Conclusions:

According to these results, the classification (1272/2008/ECC regulation) of the test item, Copper Guanylurea Nitrate (CuGUN), is the following:
- Category 4; H302: harmful if swallowed.

Executive summary:

The objective of this study was to evaluate the acute oral toxicity of the test item Copper Guanylurea Nitrate (CuGUN), in rat. The study design was based on OECD Guideline No. 423 "Acute Oral Toxicity – Acute Toxic Class Method", December 17th, 2001.

Method

In the preliminary assay, the solubility, the stability and the homogeneity of the test item in filtered tap water were assessed at 30 g/L and 200 g/L. CuGUN preparations were stirred continuously, in order to obtain and maintain a homogeneous suspension, throughout the administration.

For each dose, six nulliparous and non gravid female WISTAR rats were treated with test item in 2 successive sessions (3 rats per session). The start of the next session and/or session using the higher dose of the test item were only carried out when the certainty was obtained that the rats, treated on the preceding session, survived.

For each session, each animal was observed at least once a day for mortality, clinical signs and body weight, during 15 days. On completion of the observation period, a necropsy including macroscopic examination was realized on animals after scheduled or unscheduled death, when possible. A microscopic examination was performed on organs with macroscopic abnormality.

Results

Test item doses and treatment

At 30 g/L and 200 g/L, the test item was not soluble in water; a homogeneous suspension was obtained in filtered tap water, which was stable for at least one hour, Animals were treated at the following doses: 297 ± 4 mg/kg and 1,996 ± 21 mg/kg.

Mortality and clinical signs:

No unscheduled deaths and no clinical signs were observed on rats treated with the test item at 300 mg/kg, during the observation period. All rats treated with the test item at 2,000 mg/kg, presented an excessive salivation, for 10 or 30 minutes after treatment. Unscheduled deaths of 4 rats (1 rat in the first session and 3 rats in the second session) treated at 2,000 mg/kg were noted between day 1 and day 4. Signs of toxicity were observed until their death: a weight loss, blue liquid faeces or absence of faeces, round back, piloerection, eyelids partially closed. The two rats treated at 2,000 mg/kg, which survived during the observation period, presented blue soft faeces on day 1 and day 2.

Macroscopic and microscopic examination

Macroscopic changes considered to be related to the treatment of CuGUN were observed by external examination and in stomach, in small and large intestine, in rectum and in kidneys in rats treated with 2,000 mg/kg dose which died before the end of the study. Brown nose (2 rats), brown contour of the eyes (1 rat), soiled urogenital area (1 rat) and soiled blue green anogenital area (1 rat) were observed by external examination. Stomach presented mucosal red spots/areas (4 rats), was distended (1 rat), was filled with dark or black/blue colour content (3 rats). Small and large intestine were normal but empty of faecal matter (2 rats) and filled with black mucous (1 rat) or gray liquid content (1 rat). Some black faeces were found in rectum of one rat. Kidneys presented some dark spots (1 rat).

Microscopic changes considered to be related to treatment were restricted to stomach (3 rats), forestomach (1 rat), kidneys (1 rat) and thymus (1 rat) from rats treated with 2,000 mg/kg of CuGUN which died before the end of the study. It consisted in degeneration/necrosis/apoptosis in the stomach/forestomach, lymphocytolysis in the thymus and tubular degeneration/regeneration with crystal deposition in the kidneys.

Conclusion

According to these results, the classification (1272/2008/ECC regulation) of the test item, Copper Guanylurea Nitrate (CuGUN), is the following:

-                   Category 4; H302: harmful if swallowed.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

300 mg/kg bw

Quality of whole database:

Only one study available but quality is sufficient for LD50 determination.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-12-04 to 2013-03-04

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CHARLES RIVERS breeder, Domaine des Oncins BP 109, 69592 L’Arbresle Cedex, FRANCE
- Weight at study initiation: 238 g (female) and 246 g (male)
- Fasting period before study: no fasting period
- Housing: polycarbonate cages (Makrolon) containing autoclaved dust-free bedding (sawdust + chips), one rat per cage
- Diet: ad libitum rat/mice Altromin 1320 (Genestil, 1 rue du mesnil, 60420 ROYAUCOURT, FRANCE)
- Water: ad libitum filtered tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40%-70%
- Air changes (per hr): 15 to 20 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: december 4 2012 To: december 18 2012

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: dorsal area of the trunk
- % coverage: 10%
- Type of wrap if used: A 35 cm2-gauze dressing, corresponding to the 10% of the body surface, was placed on a saran-wrap. The test item was applied uniformly as a powder over the gauze dressing area. The test item is put in contact with the skin by applying the gauze to the area stripped of the animal. The animal was wrapped with non-irritating bandage. Care was taken to ensure bandages do not interfere with respiration and to minimize interference with animal’s movements.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, the site of treatment was rinsed and wiped delicately with water
- Time after start of exposure: 24 hours

TEST MATERIAL
- Amount(s) applied: 488 ± 12 mg of test item per rat
- For solids, paste formed: no

Duration of exposure:

24 hours

Doses:

rats were treated with 2025 ± 30 mg/kg of body weight and with 2007 ± 10 mg/kg of body weight respectively for the male and the female.

No. of animals per sex per dose:

5 males and 5 females per dose

Control animals:

not required

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations: on the day of treatment (D0): 30 minutes after the end of application, 2 times during the following 4 hours, and once thereafter. At D1, after removal of the bandage and rinsing of the skin, the site of treatment were observed carefully. Daily from D2 to D14 (once per day).
- Frequency of weighing: on day 0, before treatment and daily from D1 to D14.
- Necropsy of survivors performed: yes
- Necropsy observations: external examination (skin, especially the treatment site, eyes, nose), thoracic area (respiratory tracts, the trachea-bronchial lymph nodes, heart, and esophagus), Abdominal area (liver, spleen, the pancreas, the digestive tract, the adrenal glands, the kidneys, the bladder, the liver and kidneys lymphatic ganglia, the lumbar aortic lymphatic ganglia and female reproductive organs).

Statistics:

no statistical tests

Preliminary study:

no preliminary study

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Mortality:

no mortality.
All animals were euthanized on day 14, at the end of the study.

Clinical signs:

other: No clinical sign was observed in any of the rats during the 14 days of in-life observations.

Gross pathology:

No macroscopic abnormality was observed in animals except a partial growth back of the fur in 3 males and in the 5 females. In absence of control data, we could not conclude of any interaction between the fur growth and CuGUN application.

Interpretation of results:

study cannot be used for classification

Remarks:

Migrated information

Conclusions:

Under the experimental conditions of this study, the test item, CuGUN (lot No.40) at a dose of 2,000 mg/kg, did not cause any acute toxicity by contact with skin. Therefore, according to the regulation 1272/2008/EEC, the classification of the test item is "not classified".

Executive summary:

The objective of this study was to evaluate the acute dermal toxicity of the test item Copper Guanylurea Nitrate (CuGUN), in rat. The study design was based on OECD Guideline No. 402.

Acute dermal toxicity was determined by a limit test at one dose level of at least 2,000 mg per kg bodyweight. Because of test item related mortality was not produced, a full study was not considered.

Method

Five males and five nulliparous and non gravid female WISTAR rats were treated with test item. Test item wasapplied as a powder to the shaven skin of the animal, on a surface corresponding to 10% of the body surface. The animal was wrapped with non-irritating bandage.The test item was left in place during 24 hours. At the end of the treatment, the bandage and all dressings were removed delicately and the site of treatment was rinsed and wiped delicately with water, to ensure elimination of potential residual test item.

Each animal was observed at least once a day for mortality, clinical signs and body weight, during 15 days. On completion of the observation period, a necropsy including macroscopic examination was realized on animals after scheduled death.

Results

Rats were treated with 2,026 ± 30 mg/kg and 2,009 ± 10 mg/kg respectively for the male and the female.

No unscheduled deaths, no clinical signs and no macroscopic changes considered to be related to the treatment of CuGUN were observed on rats treated, during the observation period.

Conclusion

Under the experimental conditions of this study, the test item, CuGUN (lot No.40) at a dose of 2,000 mg/kg, did not cause any acute toxicity by contact with skin. Therefore, according to the regulation 1272/2008/EEC, the classification of the test item is "not classified".

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Only one study available but quality is sufficient for LD50 determination.

Additional information

Acute oral toxicity

·        No adverse effect observed at 300 mg/kg.

·        Target organs:stomach, thymus and kidneys.

·        Clinical signs at 2,000 mg/kg: unscheduled death of 4 rats, excessive salivation, weight loss, blue liquid faeces or absence of faeces, round back, piloerection, eyelids partially closed, blue soft faeces.

·        Macroscopic adverse effect at 2,000 mg/kg:brown nose (2 rats), brown contour of the eyes (1 rat), soiled urogenital area (1 rat) and soiled blue green anogenital area (1 rat) were observed by external examination. Stomach presented mucosal red spots/areas (4 rats), was distended (1 rat), was filled with dark or black/blue colour content (3 rats). Small and large intestine were normal but empty of faecal matter (2 rats) and filled with black mucous (1 rat) or gray liquid content (1 rat). Some black faeces were found in rectum of one rat. Kidneys presented some dark spots (1 rat).

·        Microscopic adverse effect at 2,000 mg/kg:Degeneration/necrosis/apoptosis in the stomach/forestomach, lymphocytolysis in the thymus and tubular degeneration/regeneration with crystal deposition in the kidneys.

Based on the available information, CuGUN is harmful by oral route.

Acute dermal toxicity

No unscheduled deaths, no clinical signs and no macroscopic changes.

Based on the available information, the acute toxicity of CuGUN is low for dermal route (no toxicity for the highest dose).

Justification for selection of acute toxicity – oral endpoint
The study was performed in accordance to OECD 423 and EC B.1.ter guideline, and to GLP. One deviation was observed but it was considered not to have compromised the validity or integrity of the study.

Justification for selection of acute toxicity – dermal endpoint
The study was performed in accordance to OECD 402 and EC B.3 guideline, and to GLP. No deviation was observed.

Justification for classification or non-classification

Acute oral exposure of CuGUN show a moderate toxicity for the dose of 2000 mg/kg and unscheduled death of 4 rats was observed. The LD50 is then lower than 2000 mg/kg but upper than 300 mg/kg because no adverse effect and mortality were observed for this dose. According to 1272/2008 regulation and OECD guideline 423, CuGUN must be classified as Acute Toxic category 4.

Acute dermal exposure to CuGUN shows no toxicity for the highest dose of 2000 mg/kg (LD50 > 2000 mg/kg). According to 1272/2008 regulation and OECD guideline 402, CuGUN is not classified for acute dermal toxicity.